Yuko Seko

RETINOIC ACID INCREASES IN THE RETINA OF THE CHICK WITH FORM DEPRIVATION MYOPIA

We previously reported that expression of retinoic acid receptor β increases in the sclera of the 2-week-old chick with form deprivation myopia (FDM) and that all-trans-retinoic acid (t-RA) influences proliferation and differentiation of scleral cells. The purpose of this study was to quantify t-RA in the retina of the chick with FDM and to investigate the role of t-RA in FDM in the chick. FDM was induced in 2-day-old chicks by placement of a translucent plastic goggle over one eye, with the contralateral eye used as a control. After 5 days, the chicks were sacrificed. t-RA was extracted from neural retina and served for high-performance liquid chromatography analysis. 3H-t-RA was used for normalization. Pieces of the retinae from 5 eyes served as one sample. As a result, t-RA was 0.960 ± 0.086 ng/eye (0.387 ± 0.056 ng/mg protein) in the myopic retina and 0.864 ± 0.108 ng/eye (0.319 ± 0.043 ng/mg protein) in the control retina. The concentration of t-RA (ng/mg protein) in the myopic retina was significantly higher than that in the control (p < 0.05, n = 7). These results demonstrate that t-RA increases in the retina within 5 days after visual deprivation. This finding suggests that t-RA may play a role in the metabolic changes in FDM.

INFLUENCE OF BFGF AS A POTENT GROWTH STIMULATOR AND TGF-BETA AS A GROWTH REGULATOR ON SCLERAL CHONDROCYTES AND SCLERAL FIBROBLASTS IN VITRO

We investigated the proliferation of scleral cells in response to several growth factors in vitro to elucidate the mechanism of scleral growth in visual deprivation myopia. Scleral chondrocytes and scleral fibroblasts were cultured separately in 24-well culture dishes. The number of plated cells was counted after the addition of basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGF-alpha), TGF-beta, insulin-like growth factor I (IGF-I), IGF-II, platelet-derived growth factor AA (PDGF-AA), PDGF-AB and PDGF-BB. All the growth factors studied, except for PDGF-AA, stimulated the proliferation of both scleral chondrocytes and scleral fibroblasts. bFGF showed the highest effect. TGF-beta caused morphologic changes in both scleral chondrocytes and scleral fibroblasts. Various growth factors stimulated the proliferation of scleral chondrocytes and scleral fibroblasts in a similar manner. bFGF was a potent growth stimulator and TGF-beta was suggested to be a growth regulator on scleral chondrocytes and scleral fibroblasts.

Human sclera maintains common characteristics with cartilage throughout evolution.

Background The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm. Methodology/Principal Findings We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-βs and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human ‘sclera’-derived cells can be considered chondrocytes when cultured ex vivo. Conclusions/Significance Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia.

Clinical course of newly developed or progressive patchy chorioretinal atrophy in pathological myopia

Regional chorioretinal atrophy in the posterior fundus (patchy chorioretinal atrophy) in pathological myopia impairs vision severely when it covers the macula. The aim of this study was to assess the course of development and progression of patchy chorioretinal atrophy in pathological myopia. The location and progression of patchy chorioretinal atrophy that was either newly developed or had progressed during the follow-up period (mean 5.25 years) were analyzed. A total of 41 lesions of patchy atrophy were newly developed in 30 eyes of 25 patients. These lesions were more likely to occur in marginal regions of a posterior staphyloma but frequency per unit area was highest in the macula. There were 138 lesions of patchy chorioretinal atrophy that progressed in 75 eyes of 53 patients. Sixty percent of the lesions of patchy chorioretinal atrophy in marginal regions of a posterior staphyloma spread toward the center. Seventy percent of the lesions of patchy chorioretinal atrophy in the macula spread in all directions. Fluorescein angiography of newly developed patchy chorioretinal atrophy showed hyperfluorescence in 50% and hypofluorescence in 27%. Fluorescein angiography of progressive lesions of patchy chorioretinal atrophy showed hypofluorescence in 69%. Fluorescein angiography of some progressive areas of patchy chorioretinal atrophy, which showed a change from hyperfluorescence to hypofluorescence within several years, suggested that damage to the retinal pigment epithelium preceded the progression of the patchy chorioretinal atrophy. In conclusion, the patchy chorioretinal atrophy is most likely to occur in the macula and to enlarge in all directions. And it is suggested that the patchy chorioretinal atrophy which shows hyperfluorescence by fluorescein angiography should be kept under observation because our data suggest that this finding indicates progression in the future.

Scleral cell growth is influenced by retinal pigment epithelium in vitro

Abstract• Background: Studies of form-deprivation myopia in chicks have shown that mitotic activity and the amounts of DNA and protein are increased in the sclera of myopic eyes. Signals acting on the sclera have been speculated to have their origin in the retina, especially in the photoreceptors and the retinal pigment epithelium (RPE). Therefore, in this study we investigated the interaction between the RPE cells and the scleral cells in vitro using chick embryo • Methods: At first, we established the method for primary culture of scleral chondrocytes and scleral fibroblasts from chick embryos. Then, we co-cultured scleral chondrocytes with RPE cells and also co-cultured scleral fibroblasts with RPE cells. Scleral cells were cultured on a collagen membrane or Transwell-COL, with RPE cells cultured at the bottom of the dishes. We evaluated the effects of cocultured RPE cells on the proliferation of scleral cells by counting the cell number of scleral cells on those membranes • Results: With the co-cultured RPE cells, proliferation of the scleral chondrocytes was inhibited on a collagen membrane, but significantly stimulated on a Transwell-COL. Proliferation of the scleral fibroblasts was also promoted on the Transwell-COL with RPE cells • Conclusion: RPE cells influenced the proliferation of scleral cells in vitro, which suggests that RPE may, at least in part, act on the sclera in vivo to regulate ocular growth postnatally.

Blue light-induced apoptosis in cultured retinal pigment epithelium cells of the rat.

Abstract Background: We previously demonstrated that phagosome-free retinal pigment epithelium (RPE) cells in culture can be damaged directly by blue light (wavelength 440±10 nm) as observed by electron microscope. A low intensity (1.0 mW/cm2) of light induced only swelling of mitochondria, while a high intensity (4.0 mW/cm2) induced necrosis in the RPE. The aim of the present study was to investigate what intensity of blue light could induce apoptosis in cultured phagosome-free RPE. Methods: Primary cultured RPE cells, harvested from Long-Evans rats, that contained no phagosomes were exposed to a cool blue light (wavelength 440±10 nm). After exposure, transmission electron microscopy (TEM) and TdT-mediated dUTP nick-end labeling (TUNEL) staining were used to detect apoptosis in the RPE cells. To assess the relationship of oxidation to apoptosis by blue light, we added N-acetyl-cysteine (NAC) as a free radical scavenger and investigated its inhibitory effect on apoptosis. Results: In RPE cells exposed to blue light of 2.7 mW/cm2 for 24 h, apoptotic bodies were found by TEM. In RPE cells exposed to blue light of 2.0 mW/cm2 for 60 h, apoptotic bodies, nuclear condensation and nuclear segmentation were observed by TEM and some RPE cells showed positive TUNEL staining. When 30 mM of NAC was added, TUNEL staining was negative. Conclusion: Our findings demonstrate that apoptotic cell death is induced by blue light exposure in cultured RPE cells in vitro. The findings of our previous experiments and those of the present study suggest that a higher intensity of blue light could induce necrosis, and moderately intense blue light could induce non-necrotic cell death or apoptosis, in RPE cells. Furthermore, it is suggested that blue light caused cell death by a free-radical-associated mechanism.

Derivation of Human Differential Photoreceptor-like Cells from the Iris by Defined Combinations of CRX, RX and NEUROD

Examples of direct differentiation by defined transcription factors have been provided for beta-cells, cardiomyocytes and neurons. In the human visual system, there are four kinds of photoreceptors in the retina. Neural retina and iris-pigmented epithelium (IPE) share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons. We here define the transcription factor combinations that can determine human photoreceptor cell fate. Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Phototransduction-related genes as well as opsin genes were up-regulated in those cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that generated photoreceptor cells, induced by CRX, RX and NEUROD, responded to light. The response was an inward current instead of the typical outward current. These data suggest that photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX generate immature photoreceptors: and additional NEUROD promotes maturation. These findings contribute substantially to a major advance toward eventual cell-based therapy for retinal degenerative diseases.

Observation of ultrastructural changes in cultured retinal pigment epithelium following exposure to blue light

Abstract · Background: The retina can be damaged by light even when levels of energy are well below the threshold for thermal damage, and the experimental damage of the retinal pigment epithelium (RPE) may be induced more easily by blue light than by longer wavelengths of visible light. The present study demonstrates the ultrastructural damage produced by exposure to blue light in cultured RPE. · Methods: Long-Evans rats were enucleated 8–10 days after birth for primary culture. One week after seeding, the monolayer culture of RPE cells was exposed to a cool blue light (wavelength = 440±10 nm) for 36 h (12 h/day, 3 days) at 2.0 mW/cm2. Transmission electron microscopy was used to compare the exposed RPE with the control. The entire experiment was repeated 3 times independently. · Results: The cytoplasm of the exposed RPE exhibited degenerative changes, such as large whorls of membrane, lamellar whorls and whorled inclusions. · Conclusion: The RPE cells can be damaged directly by blue light after excluding the possible influence of phagosomes. This primary culture of RPE can also serve as an in vitro model for the study of light damage to the RPE.

Establishment of functioning human corneal endothelial cell line with high growth potential.

Hexagonal-shaped human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na+- and K+-dependent ATPase (Na+/K+-ATPase). Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs) in the Rb pathway (p16-CDK4/CyclinD1-pRb). In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7)) and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin)). Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7), THCEH (Cyclin) and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7) and THCEH (Cyclin). THCEH (Cyclin) expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na+/K+-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7). This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.

Disturbance of Electrolyte Balance in Vitreous of Chicks With Form-Deprivation Myopia

PURPOSE To investigate the changes in the electrolyte and protein concentrations in the vitreous of 3-week-old chicks with form-deprivation myopia (FDM). METHODS FDM was induced in 2-day-old male white leghorn chicks by covering the left eye with a translucent plastic goggle and leaving the right eye uncovered to serve as control. After 19 days the animals were euthanized, and the axial dimensions of the eyes were measured with a caliper in an unfixed condition. The liquid vitreous and aqueous humor were removed by paracentesis, and blood was collected from the jugular vein. Sodium, potassium, and chloride concentrations were determined using ion-selective electrodes. Calcium and phosphate concentrations were determined by colorimetric assays using orthocresol phthalein complexone and bacterial xanthine oxidase, respectively. RESULTS The concentrations of potassium and phosphate were decreased, whereas chloride concentration was increased in the vitreous of the FDM eyes (P < .01). Sodium and calcium concentrations were similar to those in the control eyes. No significant changes in the concentration of electrolytes were observed in the aqueous humor. No significant differences were found in the protein concentrations in the liquid vitreous, gel vitreous, and aqueous humor. CONCLUSIONS Form-deprivation induced a significant increase of the volume of the liquid vitreous in the eye of the FDM chick. The increased liquid vitreous of the myopic eye was accompanied by an alteration of the electrolyte balance, by a mechanism that has not yet been clarified.